Sox2 regulates Müller glia reprogramming and proliferation in the regenerating zebrafish retina via Lin28 and Ascl1a

Abstract

Sox2 is a well-established neuronal stem cell-associated transcription factor that regulates neural development and adult neurogenesis in vertebrates, and is one of the critical genes used to reprogram differentiated cells into induced pluripotent stem cells. We examined if Sox2 was involved in the early reprogramming-like events that Müller glia undergo as they upregulate many pluripotency- and neural stem cell-associated genes required for proliferation in light-damaged adult zebrafish retinas. In the undamaged adult zebrafish retina, Sox2 is expressed in Müller glia and a subset of amacrine cells, similar to other vertebrates. Following 31 hours of light damage, Sox2 expression significantly increased in proliferating Müller glia. Morpholino-mediated knockdown of Sox2 expression resulted in decreased numbers of proliferating Müller glia, while induced overexpression of Sox2 stimulated Müller glia proliferation in the absence of retinal damage. Thus, Sox2 is necessary and sufficient for Müller glia proliferation. We investigated the role of Wnt/β-catenin signaling, which is a known regulator of sox2 expression during vertebrate retinal development. While β-catenin 2, but not β-catenin 1, was necessary for Müller glia proliferation, neither β-catenin paralog was required for sox2 expression following retinal damage. Sox2 expression was also necessary for ascl1a (neurogenic) and lin28a (reprogramming) expression, but not stat3 expression following retinal damage. Furthermore, Sox2 was required for Müller glial-derived neuronal progenitor cell amplification and expression of the pro-neural marker Tg(atoh7:EGFP). Finally, loss of Sox2 expression prevented complete regeneration of cone photoreceptors. This study is the first to identify a functional role for Sox2 during Müller glial-based regeneration of the vertebrate retina.

Midkine-a is required for cell cycle progression of Müller glia during neuronal regeneration

“…As a result, the Müller glia-derived regeneration of cone photoreceptors fails, but rod photoreceptors are eventually replenished. This observation is consistent with previous reports that also identified separate lineages between regenerated cone and rod photoreceptors (Gorsuch et al, 2017;Morris et al, 2008;Thummel et al, 2010).…”

“…In response to neuronal cell death, Müller glia spontaneously reprogram, upregulating stem cellassociated genes prior to entering the cell cycle (Goldman, 2014;Gorsuch and Hyde, 2014;Hamon et al, 2016;Lenkowski and Raymond, 2014). Immunostaining retinas at 1 and 2 dpl for the stem-cell associated proteins, Rx1 and Sox2, labeled elongated, polygonal nuclei, characteristic of Müller glia (Nagashima et al, 2013) (Gorsuch et al, 2017), in both wildtype and mutant retinas (Fig. 4A).…”

Cross-species transcriptomic and epigenomic analysis reveals key regulators of injury response and neuronal regeneration in vertebrate retinas

“…Reactive gliosis is a critical step in the formation of MGPCs, as loss of function of genes that are highly enriched in reactive MG such as hmga1a in zebrafish and FABP family genes in chick blocks the formation of MGPCs. Previous studies in zebrafish have likewise identified multiple genes that show enriched expression in reactive MG --such as six3b, sox2, lepb and others (25,35,45) --to also be essential for MGPC formation.…”

DNA demethylation is a driver for chick retina regeneration

“…TET1-2 are important for ESC linage specification [93,94]. TET1 plays an important role during DNA demethylation and gene expression in mouse ES cells [86][87][88]95] while TET3 contributes to genome-wide DNA demethylation and can efficiently reprogram fibroblast directly into functional neurons and induce demethylation of Ascl1, an important factor for MG reprogramming in mouse and zebrafish [96][97][98][99][100][101][102]. Moreover, TET3 is required for significant axon regeneration in the dorsal root ganglion (DRG) neurons, suggesting that an epigenetic barrier can be removed by active DNA demethylation mediated by TET3 [103].…”